Osteoarthritis (OA) is painful and debilitating by affecting the synovial joints, and is found in over 12% of the total United States population 25-74 years of age. The prevalence of OA increases significantly with age, with radiographic evidence in over 70% of the population over age 65. In this growing segment of our society, OA is a significant contributor to disability, frailty and social isolation. Despite the tremendous socioeconomic impact of OA, there are no disease-modifying therapies available. OA is distinctively characterized by the progressive, degenerative changes in the morphology, composition, and mechanical properties of articular cartilage. Mechanotransduction in articular chondrocytes is a key component of disease pathogenesis, given the link between direct sensing of the cells? mechanical environment and the resulting metabolic imbalance of cartilage in OA. We have recently identified the mechanosensitive PIEZO ion channels - in fact a synergy between PIEZO1 and PIEZO2, both expressed in articular cartilage - to underlie chondrocyte mechanotransduction in response to injurious mechanical stress. The overall objective of this study is to define the mechanisms of Piezo-mediated mechanotransduction in chondrocytes more in-depth so that these insights can be leveraged toward the development of disease- modifying approaches in joint-loading-induced injuries, including OA. In addition to our recent discovery of chondrocytic Piezo-mediated mechanotransduction, we found that treatment of chondrocytes with pathophysiologically-relevant concentrations of IL-1?, a pro-inflammatory cytokine, increased Piezo1 gene expression, and that increased expression of Piezo1 was also present in osteoarthritic cartilage from aging pigs and humans. Thus, the Specific Aims of this grant are: (1) to determine the mechanisms of synergistic functioning of Piezo1/2 in chondrocyte mechanotransduction; (2) to deconstruct Piezo-mediated mechanotransduction in chondrocytes under inflammatory conditions; (3) to elucidate the role of Piezo- mediated mechanotransduction in organotypic cartilage explants and in-vivo. Aim 1 will rely on cellular studies. We will explore synergisms of Piezo1/2 at the levels of electrophysiology, channel trafficking, finite element modeling, and ultra-structure, the latter also examining human cartilage from OA vs controls. In Aim 2 primary porcine chondrocytes will be stimulated with IL-1? for deconstruction of Piezo-mediated mechanotransduction. Aim 3 will rely on porcine osteochondral explants and chondrocyte-specific and inducible Piezo1/2-/- mice which we have generated. Various modes of mechanical stress will be applied to cells, explants, and animals, and loss-of-function studies of Piezo-mediated mechanotransduction will be conducted with both mechanistic intent and translational/therapeutic direction. The proposed Aims will extend our initial discovery with mechanistic in- depth studies that will increase our understanding of OA in a non-incremental manner, and this will inspire the development of new Disease-Modifying OA Drugs (DMOADs).